The implementation of high-throughput technologies based on qualitative and quantitative methodologies for the characterization of complex protein mixtures is increasingly required in clinical laboratories. To this purpose, MALDI profiling appears a robust and sensitive technology even though the high dynamic range imposes a major limitation a hampering the identification of less abundant species and decreasing the quality of MALDI profiling for clinical implementations. A number of methodologies have been developed to overcome this issue and in this study the efficacy and reproducibility of immunodepletion for human serum profiling was investigated. Two commercially available columns (MARS Hu7 and Hu14) were tested. The effects of both on serum were assessed by 2D-DIGE: the comparison of eluted fraction from Hu14 versus Hu7 revealed the presence of a different protein distribution and of 128 differentially changed spots (p-value<0.01), out of them 114 were identified by mass spectrometry. In particular, 34 spots strongly decreased in Hu14 sample correspond to proteins and their isoforms expected to be significantly reduced by this column. In addition, the type of matrix and the sample dilution, representing crucial elements for MALDI profiling improvement, were determined. Finally, the selected experimental conditions were applied to sera profiling of 8 subjects before and after erythropoietin treatment. The results indicate that a combination of Hu14 column immunodepletion and DHAP matrix increases data quality and appears an appropriate approach for human serum profiling by MALDI.
Set-up for MALDI profiling of human sera: the case of rhEPO teatment / M. Vasso, C. Fania, E. Torretta, C. Gelfi. ((Intervento presentato al convegno Proteomic Forum tenutosi a Berlin nel 2011.
|Titolo:||Set-up for MALDI profiling of human sera: the case of rhEPO teatment|
VASSO, MICHELE (Primo)
FANIA, CHIARA (Secondo)
TORRETTA, ENRICA (Penultimo)
GELFI, CECILIA (Ultimo)
|Data di pubblicazione:||2011|
|Settore Scientifico Disciplinare:||Settore BIO/12 - Biochimica Clinica e Biologia Molecolare Clinica|
|Citazione:||Set-up for MALDI profiling of human sera: the case of rhEPO teatment / M. Vasso, C. Fania, E. Torretta, C. Gelfi. ((Intervento presentato al convegno Proteomic Forum tenutosi a Berlin nel 2011.|
|Appare nelle tipologie:||14 - Intervento a convegno non pubblicato|